U.S. patent application number 16/102997 was filed with the patent office on 2019-05-02 for nucleic acid probes and methods for detecting plasmodium parasites.
This patent application is currently assigned to ID-Fish Technology, Inc.. The applicant listed for this patent is ID-Fish Technology, Inc.. Invention is credited to Nick S. Harris, Oliva Mark, Suzanne Scherini-Ward, Jytosna S. Shah, Helena Weltman, Danuta Wronska.
Application Number | 20190127809 16/102997 |
Document ID | / |
Family ID | 39468504 |
Filed Date | 2019-05-02 |
United States Patent
Application |
20190127809 |
Kind Code |
A1 |
Shah; Jytosna S. ; et
al. |
May 2, 2019 |
NUCLEIC ACID PROBES AND METHODS FOR DETECTING PLASMODIUM
PARASITES
Abstract
This invention relates to novel nucleic acid probes and methods
for detecting Plasmodium parasites as well as detecting different
Plasmodium parasites selectively from one another.
Inventors: |
Shah; Jytosna S.; (Santa
Clara, CA) ; Wronska; Danuta; (Raleigh, NC) ;
Weltman; Helena; (Los Altos, CA) ; Harris; Nick
S.; (Monterey, CA) ; Scherini-Ward; Suzanne;
(Santa Clara, CA) ; Mark; Oliva; (San Jose,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ID-Fish Technology, Inc. |
Santa Clara |
CA |
US |
|
|
Assignee: |
ID-Fish Technology, Inc.
Santa Clara
CA
|
Family ID: |
39468504 |
Appl. No.: |
16/102997 |
Filed: |
August 14, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15247305 |
Aug 25, 2016 |
10077480 |
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16102997 |
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14060215 |
Oct 22, 2013 |
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15247305 |
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13085799 |
Apr 13, 2011 |
8592568 |
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14060215 |
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11998251 |
Nov 29, 2007 |
7927801 |
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13085799 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/30 20180101;
C12Q 1/6893 20130101; C12Q 2600/16 20130101; Y02A 50/58
20180101 |
International
Class: |
C12Q 1/6893 20060101
C12Q001/6893 |
Claims
1. A method for detecting the presence of Plasmodium malariae in a
biological sample, said method comprising the steps of: a)
providing a biological sample to be analyzed for the presence of
Plasmodium malariae; b) providing a composition suitable for
detecting Plasmodium malariae in the sample, said composition
selected from one or more of: a nucleic acid sequence consisting of
a labeled [SEQ ID NO: 14] or the labeled full-length complementary
sequence thereof; c) contacting said sample with the composition
suitable for detecting Plasmodium malariae in a hybridization assay
under conditions that permit said probe to hybridize to Plasmodium
malariae nucleic acid; and b) detecting said probe bound to said
Plasmodium malariae nucleic acid in said sample as an indication of
the presence of Plasmodium malariae in said sample.
2. The method of claim 1, wherein said nucleic acid probe is a
nucleic acid sequence consisting of a labeled [SEQ ID NO: 14].
3. The method of claim 1, wherein said nucleic acid probe is a
labeled full-length complementary sequence of [SEQ ID NO: 14]
thereof.
4. The method of claim 1, wherein said detecting comprises
detecting probe bound to said Plasmodium malariae nucleic acid by
Fluorescent in Situ Hybridization (FISH).
5. The method of claim 1, wherein said detecting comprises
detecting probe bound to said Plasmodium malariae nucleic acid by a
Dot Blot assay.
6. A composition comprising the nucleotide sequence consisting of
[SEQ ID NO: 14], wherein the nucleotide sequence is labeled.
7. A composition comprising the complementary sequence to the
nucleotide sequence consisting of [SEQ ID NO: 14], wherein the
nucleotide sequence is labeled.
8. A method for detecting the presence of Plasmodium ovale in a
sample, said method comprising the steps of: a) providing a
biological sample to be analyzed for the presence of Plasmodium
ovale; b) providing a composition suitable for detecting Plasmodium
ovale in the sample, said composition selected from one or more of:
a nucleic acid sequence consisting of a labeled [SEQ ID NO: 15] or
the labeled full-length complementary sequence thereof; c)
contacting said sample with the composition suitable for detecting
Plasmodium ovale in a hybridization assay under conditions that
permit said probe to hybridize to Plasmodium ovale nucleic acid;
and b) detecting said probe bound to said Plasmodium ovale nucleic
acid in said sample as an indication of the presence of Plasmodium
ovale in said sample.
9. The method of claim 8, wherein said nucleic acid probe is a
nucleic acid sequence consisting of a labeled [SEQ ID NO: 15].
10. The method of claim 8, wherein said nucleic acid probe is a
labeled full-length complementary sequence of [SEQ ID NO: 15]
thereof.
11. The method of claim 8, wherein said detecting comprises
detecting probe bound to said Plasmodium ovale nucleic acid by
Fluorescent in Situ Hybridization (FISH).
12. The method of claim 8, wherein said detecting comprises
detecting probe bound to said Plasmodium ovale nucleic acid by a
Dot Blot assay.
13. A composition comprising the nucleotide sequence consisting of
[SEQ ID NO: 15], wherein the nucleotide sequence is labeled.
14. A composition comprising the complementary sequence to the
nucleotide sequence consisting of [SEQ ID NO: 15], wherein the
nucleotide sequence is labeled.
Description
BACKGROUND OF THE INVENTION
[0001] Plasmodium is a genus of parasitic protozoa. Infection with
this genus is known as malaria. The parasite always has two hosts
in its life cycle: a mosquito vector and a vertebrate host. At
least ten species infect humans including P. falciparum, P. vivax,
P. malariae, and P. ovale. Malaria is an infectious disease that is
widespread in tropical and subtropical regions. Malaria represents
a threat to survival of men, women and children. It infects between
300 and 500 million people every year and causes between one and
three million deaths annually, mostly among young children in
Sub-Saharan Africa.
[0002] Malaria is one of the most common infectious diseases and an
enormous public-health problem. The disease is caused by protozoan
parasites of the genus Plasmodium. The most serious forms of the
disease are caused by Plasmodium falciparum and Plasmodium vivax,
but other related species (Plasmodium ovale and Plasmodium
malariae) can also infect humans. Determining the infectious
species helps determine the course of treatment for the
patient.
[0003] The preferred and most reliable diagnosis of malaria is
microscopic examination of blood films because each of the four
major parasite species has distinguishing characteristics. Two
sorts of blood film are traditionally used. Thin films are similar
to usual blood films and allow species identification because the
parasite's appearance is best preserved in this preparation. Thick
films allow the microscopist to screen a larger volume of blood and
are about eleven times more sensitive than the thin film, so
picking up low levels of infection is easier on the thick film, but
the appearance of the parasite is much more distorted and therefore
distinguishing between the different species can be much more
difficult.
[0004] From the thick film, an experienced microscopist can detect
parasite levels (or parasitemia) down to as low as 0.0000001% of
red blood cells. However, microscopic diagnosis can be difficult
because the early trophozoites ("ring form") of all four species
look identical and it is never possible to diagnose species on the
basis of a single ring form; species identification is always based
on several trophozoites.
[0005] In areas where microscopy is not available, there are
antigen detection tests that require only a drop of blood.
OptiMAL-IT.RTM. (TCS Bio Sciences, Buckingham, England) will
reliably detect falciparum down to 0.01% parasitemia and
non-falciparum down to 0.1%. Paracheck-Pf.RTM. (Orchard Biomedical
Systems, India) will detect parasitemias down to 0.002% but will
not distinguish between falciparum and non falciparum malaria.
Parasite nucleic acids may also be detected using polymerase chain
reaction. This technique is more accurate than microscopy. However,
it is expensive and requires a specialized laboratory. Moreover,
levels of parasitemia are not necessarily correlative with the
progression of disease, particularly when the parasite is able to
adhere to blood vessel walls. Limited molecular methods are
available in some clinical laboratories and rapid real-time assays,
for example, QT-NASBA (real-time quantitative nucleic acid
sequence-based amplification) based on the polymerase chain
reaction) but are only now being developed. Therefore, sensitive,
low-tech diagnosis tools need to be developed for in order to
detect low levels of parasitaemia in the field.
[0006] What is need are reagents and methods for the rapid and
accurate detection and discrimination of various malaria causing
Plasmodium species.
SUMMARY OF THE INVENTION
[0007] This invention relates to nucleic acid probes that detect
and discriminate between different species of Plasmodium parasites
in, for example, hybridization assays. Accordingly, in a first
aspect, the invention features nucleic acid fragments to be used as
probes for detecting Plasmodium in a hybridization assay. The
invention also includes probes (DNA, RNA and PNA) that can
discriminate between P. falciparum, P. vivax, P. malariae and P.
ovale. In the context of the present invention the term
"discriminates between" (or similar terms) means that the probe
binds to nucleic acid (e.g., RNA, DNA, rRNA [ribosomal RNA] or rDNA
[ribosomal DNA]) from one species more favorably than the other 3
species.
[0008] In a second aspect, the invention features a nucleic acid
fragment containing a sequence selected from, preferably, at least
five, more preferably at least ten or most preferably at least
fifteen consecutive nucleotides or the entire sequence of one or
more of probes designated PGenus1, PGenus2, Mal F1, MalF2, Mal1.8F,
Mal1.8R, PV1, PV2, PF1, PF2, PF3, PF4, PF5, PM1, PO1 or the partial
or full-length complementary sequences thereof.
[0009] In a third aspect, the invention features a nucleic acid
fragment containing a sequence selected from at least five,
preferably, at least ten, more preferably at least thirteen or most
preferably at least fifteen consecutive nucleotides or the entire
sequence of one or more of probes designated SEQ ID NO: 1 through
SEQ ID NO.: 15 or the partial or full-length complementary
sequences thereof.
[0010] In a final aspect, the invention features a method for
detecting the presence of Plasmodium in a sample. In this method, a
sample is contacted with a nucleic acid fragment containing a
sequence selected from, preferably, at least five, at least ten,
more preferably at least thirteen or most preferably at least
fifteen consecutive nucleotides or the entire sequence of a
PGenus1, PGenus2, Mal F1, MalF2, Mal1.8F, Mal1.8R, PV1, PV2, PF1,
PF2, PF3, PF4, PF5, PM1 or PO1, probe selected from or the partial
or full-length complementary sequence thereof (or any combination
thereof); under conditions that permit the nucleic acid fragment to
hybridize to Plasmodium nucleic acid. Detection of the nucleic acid
fragment bound to the Plasmodium nucleic acid in the sample is used
as an indication of the presence of Plasmodium in the sample.
Detection with probes of the present invention that discriminate
between the four species of Plasmodium listed above indicates the
presence of that species of Plasmodium.
[0011] In one embodiment of this aspect of the invention, the
nucleic acid fragment contains a sequence selected, preferably,
from at least five, more preferably at least ten or most preferably
at least fifteen consecutive nucleotides or the entire sequence of
probe PGenus1 or the full-length complementary sequence
thereof.
[0012] In a second embodiment of this aspect of the invention, the
nucleic acid fragment contains a sequence selected, preferably,
from at least five, more preferably at least ten or most preferably
at least fifteen consecutive nucleotides or the entire sequence of
probe PGenus2, or the full-length complementary sequence
thereof.
[0013] In a third embodiment of this aspect of the invention, the
nucleic acid fragment contains a sequence selected from,
preferably, at least five, more preferably at least ten or most
preferably at least fifteen or the entire sequence of probe MalF1
or the full-length complementary sequence there of.
[0014] In yet another embodiment of this aspect of the invention,
the nucleic acid fragment contains a sequence selected from,
preferably, at least five, more preferably at least ten or most
preferably at least fifteen nucleotides, or the entire sequence of
probe MalF2 or the full-length complementary sequence thereof.
[0015] In yet another embodiment of this aspect of the invention,
the nucleic acid fragment contains a sequence selected from,
preferably, at least five, more preferably at least ten or most
preferably at least fifteen nucleotides or the entire sequence of
probe Mal1.8F or the full-length complementary sequence
thereof.
[0016] In yet another embodiment of this aspect of the invention,
the nucleic acid fragment contains a sequence selected from,
preferably, at least five, more preferably at least ten or most
preferably at least fifteen nucleotides or the entire sequence of
probe Mal1.8R, or the full-length complementary sequence
thereof.
[0017] An advantage of probes PGenus 1, PGenus2, MalF1, MAlF2,
Mal1.8F and Mal1.8R is that while they detect all three species of
Plasmodium tested and, thus, are not limited to detecting a single
Plasmodium species. Other features and advantages of the present
invention will be apparent from the following detailed description
thereof and also from the appended claims.
[0018] In specific aspects, the present invention contemplates a
method for detecting the presence of Plasmodium in a sample, said
method comprising the steps of: contacting said sample with a probe
for detecting Plasmodium in a hybridization assay, wherein said
probe discriminates between Plasmodium species, under conditions
that permit said probe to hybridize to Plasmodium nucleic acid; and
detecting said probe bound to said Plasmodium nucleic acid in said
sample as an indication of the presence of Plasmodium in said
sample.
[0019] In the preceding embodiment, the nucleic acid fragment
comprises a sequence that is selected from at least five
consecutive nucleotides of probe PV1, PV2, PF1 PF3, PF4, PF5, PM1
or PO1 the full-length complementary sequence thereof.
[0020] In other aspects, the present invention contemplates a
method for detecting the presence of Plasmodium in a sample, said
method comprising the steps of: contacting said sample with a
nucleic acid fragment comprising a sequence selected from at least
five consecutive nucleotides of a probe selected from a group
consisting of PGenus 1, PGenus2, MalF1, MalF2, Mal1.8F, Mal1.8R,
PV1, PV2, PF1, PF2, PF3, PF4, PF5, PM1 and PO1 or full-length
complimentary sequence thereof, under conditions that permit said
nucleic acid fragment to hybridize to Plasmodium nucleic acid; and
detecting said nucleic acid fragment bound to said Plasmodium
nucleic acid in said sample as an indication of the presence of
Plasmodium in said sample.
[0021] In the preceding embodiment, the nucleic acid fragment
comprises of a sequence selected from the full length sequence, at
least fifteen consecutive nucleotides, at least ten consecutive
nucleotides, at least five consecutive nucleotides of a probe
selected from a group consisting of PGenus1, PGenus2, MalF1, MalF2,
Mal1.8F, Mal1.8R, PV1, PV2, PF1, PF2, PF3, PF4, PF5, PM1 and PO1 or
full-length complimentary sequence thereof.
[0022] The present invention also contemplates a nucleic acid
fragment comprising a sequence selected from at least five
consecutive nucleotides of a probe selected from a group consisting
of PGenus1, PGenus2, MalF1 and MalF2, Mal1.8F, Mal1.8R, PV1, PV2,
PF1, PF2, PF3, PF4, PF5, PM1, PO1 or the full-length complementary
sequence thereof.
[0023] The present invention also contemplates a nucleic acid
fragment comprising a sequence selected from at least ten
consecutive nucleotides of a probe selected from a group consisting
of PGenus1, PGenus2, MalF1 and MalF2, Mal1.8F, Mal1.8R, PV1, PV2,
PF1, PF2, PF3, PF4, PF5, PM1, PO1 or the full-length complementary
sequence thereof.
[0024] The present invention also contemplates a nucleic acid
fragment comprising the complete sequence selected of a probe
selected from a group consisting of PGenus1, PGenus2, MalF1 and
MalF2, Mal1.8F, Mal1.8R, PV1, PV2, PF1, PF2, PF3, PF4, PF5, PM1,
PO1 or the full-length complementary sequence thereof.
[0025] In specific aspects, the present invention contemplates a
method of selecting nucleic acid probes that discriminate between
the species P. falciparum, P. vivax, P. malariae and P. ovale, the
method comprising: preparing a nucleic acid fragment or polypeptide
nucleic acid, PNA corresponding to, or complementary to, a sequence
of at least five nucleotides of nucleic acid from P. falciparum, P.
vivax, P. malariae and P. ovale; comparing the ability of the probe
to detect one or more of the Plasmodium species in a hybridization
assay; and selecting the probe or probes that detect one, two or
three species of Plasmodium but not all four species of
Plasmodium.
[0026] The present invention also contemplates a method for
detecting and differentiating between P. falciparum, P. Vivax, P.
malariae and P. ovale in a sample, said method comprising:
providing: i) a sample from a subject suspected of having malaria
and ii) probes comprising nucleic acid suitable for detecting and
differentiating between P. falciparum, P. Vivax, P. malariae and P.
ovale; contacting said sample with said probes under conditions
suitable for hybridization of said probes to targets; and
determining the presence of P. falciparum, P. Vivax, P. malariae
and P. ovale, if any, in the sample.
[0027] In one embodiment, the method contemplates that the probe
for detecting P. falciparum is selected from a nucleic acid
sequence of at least five contiguous nucleotides of one or more of
PF1, PF2, PF3, PF4 or PF5. In another embodiment, the method
contemplates that the probe is selected from a nucleic acid
sequence of at least ten contiguous nucleotides of one or more of
PF1, PF2, PF3, PF4 or PF5. In yet another embodiment, the method
contemplates that the probe is selected from a nucleic acid
sequence of at least fifteen contiguous nucleotides of one or more
of PF1, PF2, PF3, PF4 or PF5.
[0028] In one embodiment, the method contemplates that the probe
for detecting P. vivax is selected from a nucleic acid sequence of
at least five contiguous nucleotides of one or more of PV1 or PV2.
In another embodiment, the method contemplates that the probe is
selected from a nucleic acid sequence of at least ten contiguous
nucleotides of one or more of PV1 or PV2. In yet another
embodiment, the method contemplates that the probe is selected from
a nucleic acid sequence of at least fifteen contiguous nucleotides
of one or more of PV1 or PV2.
[0029] In one embodiment, the method contemplates that the probes
for detecting P. malariae are selected from a nucleic acid sequence
of at least five contiguous nucleotides of PM1 and one or more of
PF1, PF2, PF3, PF4 or PF5 and wherein a sample is positive for P.
malariae if the probe of at least five contiguous nucleotides of
PM1 tests positive and the probe of at least five contiguous
nucleotides of PF1, PF2, PF3, PF4 or PF5 tests negative. In another
embodiment, the method contemplates that the probes are selected
from a nucleic acid sequence of at least ten contiguous nucleotides
of one or more of PF1, PF2, PF3, PF4 or PF5 and at least ten
contiguous nucleotides of PM1. In yet another embodiment, the
method contemplates that the probes are selected from a nucleic
acid sequence of at least fifteen contiguous nucleotides of one or
more of PF1, PF2, PF3, PF4 or PF5 and at least fifteen contiguous
nucleotides of PM1.
[0030] In one embodiment, the method contemplates that the probes
for detecting P. ovale are selected from a nucleic acid sequence of
at least five contiguous nucleotides of PO1 and one or more of PF1,
PF2, PF3, PF4 or PF5 and wherein a sample is positive for P. ovale
if the probe of at least five contiguous nucleotides of PO1 tests
positive and the probe of at least five contiguous nucleotides of
PF1, PF2, PF3, PF4 or PF5 tests negative. In another embodiment,
the method contemplates that the probes are selected from a nucleic
acid sequence of at least ten contiguous nucleotides of one or more
of PF1, PF2, PF3, PF4 or PF5 and at least ten contiguous
nucleotides of PO1. In yet another embodiment, the method
contemplates that the probes are selected from a nucleic acid
sequence of at least fifteen contiguous nucleotides of one or more
of PF1, PF2, PF3, PF4 or PF5 and at least fifteen contiguous
nucleotides of PO1.
[0031] In any of the preceding embodiments, the probe or probes
used may be of the entire nucleotide sequence as disclosed herein
or the complementary strand thereof as well as the complementary
sequence of the five, ten or fifteen contiguous nucleotides of the
probes.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The invention features nucleic acid probes for detecting
Plasmodium parasites (e.g., P. falciaprum, P. vivax, P. malariae
and P. ovale) in, for example, hybridization assays. The probes of
the invention may be used in methods for detecting the presence of
Plasmodium in a biological sample. In these methods, a probe of the
invention is contacted with a biological sample (e.g., whole blood,
CSF or a tissue sample) in a hybridization assay and detection of
the probe bound to the nucleic acid in the sample is used as an
indication of the presence of Plasmodium in the sample. Probes
included in the invention may be identified by:
[0033] A (1) preparing a nucleic acid fragment or polypeptide
nucleic acid, PNA (i.e., a probe) corresponding to, or
complementary to, a sequence of at least ten nucleotides of nucleic
acid from P. falciparum and (2) comparing the ability of the probe
to detect all the Plasmodium species in a hybridization assay.
Probes that hybridize to P. falciparum more favorably than to other
three species are included in the invention.
[0034] B (1) preparing a nucleic acid fragment or PNA (i.e., a
probe) corresponding to, or complementary to, a sequence of at
least ten nucleotides of nucleic acid from P. vivax and (2)
comparing the ability of the probe to detect all the Plasmodium
species in a hybridization assay. Probes that hybridize to P. vivax
more favorably than to other three species are included in the
invention.
[0035] C (1) preparing a nucleic acid fragment or PNA (i.e., a
probe) corresponding to, or complementary to, a sequence of at
least ten nucleotides of nucleic acid from P. malariae and (2)
comparing the ability of the probe to detect all the Plasmodium
species in a hybridization assay. Probes that hybridize to P.
malariae more favorably than to other three species are included in
the invention.
[0036] D (1) preparing a nucleic acid fragment or PNA (i.e., a
probe) corresponding to, or complementary to, a sequence of at
least ten nucleotides of nucleic acid from P. ovale and (2)
comparing the ability of the probe to detect all the Plasmodium
species in a hybridization assay. Probes that hybridize to P. ovale
more favorably than to other three species are included in the
invention.
[0037] P. falciaprum, P. vivax, P. malariae and P. ovale nucleic
acid may be obtained from biological samples (such as whole blood,
bone marrow, CSF) from infected individuals, using standard nucleic
acid isolation methods in the art. P. falciaprum (as well as P.
vivax, P. malariae and P. ovale) can also be obtained from culture.
For example, DNA encoding Plasmodium ribosomal RNA may be obtained
by PCR amplification of DNA prepared from a whole blood sample of
an infected patient using the methods and primers described herein
and known in the art.
[0038] Any Plasmodium sequence (e.g., a sequence encoding 58, 5.8S,
18S, or 28S ribosomal RNA) may be selected as a candidate sequence
for the identification of probes. Preferred sequences are those
that diverge from analogous sequences in non-human Plasmodium or
other protozoan parasites like, for example, Babesia or Thileria,
as determined by phylogenetic comparison. The nucleic acid probes
of the invention are at least 10 nucleotides in length and may
contain deoxyribonucleotides (DNA probes), ribonucleotides (RNA
probes), peptide nucleic acid (PNA probes) or combinations or
modifications thereof. The probes may be single stranded or double
stranded and may be prepared by any of a number of standard methods
in the art. For example, the probes may be made by chemical
synthesis, restriction endonuclease digestion of a vector (e.g., a
plasmid containing a sequence corresponding to the probe),
polymerase chain reaction (PCR) amplification, or in vitro
transcription of a vector containing a sequence corresponding to
the probe (see, e.g., Ausubel, et al., Current Protocols in
Molecular Biology, Greene Publishing, New York, N.Y., 1994,
incorporated herein by reference). The probes may be labeled during
or after synthesis. For example, labeled nucleotides containing,
e.g., radioisotopes (e.g., p32, S35, or H3), biotin or digoxigenin
may be incorporated into the probe during synthesis. Probes
containing biotin are detected by the use of a secondary reagent
such as avidin or streptavidin, which contains a detectable label
such as a fluorochrome (e.g., fluorescein or rhodamine) or an
enzyme (e.g., alkaline phosphatase or horseradish peroxidase).
Similarly, probes containing digoxigenin may be detected by using a
labeled antidigoxigenin antibody. Probes may also be labeled after
synthesis by, e.g., nick translation or the use of T4 RNA ligase,
poly(A) polymerase, terminal transferase or T4 polynucleotide
kinase, in standard methods (see, e.g., Ausubel, et al., supra).
The probes may also contain modified nucleotides in order to
increase the stability of the probe. For example, ribonucleotides
containing 2'-O-alkyl groups on the ribose group may be used. The
probes may also contain modifications that facilitate capture of
the probe onto a solid support. For example, poly-dA or
poly-deaza-guanosine tails may be added to the 3' ends of the
probes, using terminal transferase, in order to facilitate probe
binding to a solid support, e.g., poly-dT or poly-dC labeled
magnetic particles. The probes may be purified prior to use, using
standard methods such as denaturing polyacrylamide gel
electrophoresis, high performance liquid chromatography or gel
filtration chromatography (see, e.g., Ausubel, et al., supra). The
probes of the invention may be used in any standard hybridization
assay to detect the presence of Plasmodium in a sample. For
example, Southern blot, dot blot, in situ hybridization, real-time
hybridization detection by biosensors or dual probe, sandwich-type
hybridization assays may be used (see, e.g., U.S. patent
application Ser. No. 07/826,657 [now U.S. Pat. No. 5,519,127] and
U.S. Pat. No. 5,629,156 [International Publication Number WO
94/10335], all of which are incorporated herein by reference).
Alternatively, the probes may be used as primers in a polymerase
chain reaction assay (see, e.g., Ausubel, et al., supra).
Biological samples that may be analyzed using the probes and
methods of the invention include whole blood, CSF, bone marrow and
tissue samples from, e.g., the spleen. Nucleic acid is extracted
from the sample by standard methods (except in the case of in situ
hybridization, where the cells are kept intact) and is analyzed
using the probes in the assays listed above. A single probe or
combinations of probes may be used in the assay. The hybridization
conditions used with the probes (e.g., in the methods of the
invention) fall within the range of, for example, 30-50 formamide
at 25.degree. C.-42.degree. C. or mixtures of GuSCN and formamide
between 25-37.degree. C. As is "known by one skilled in the art,"
selection of hybridization conditions depends on the length and
nucleotide content (i.e., GC compared to AT) of the probe.
Accordingly, hybridization conditions may be adjusted to
accommodate these factors. In addition, the use of different salts
(e.g., guanidine thiocyanate or guanidine hydrochoride compared
with NaCl) and denaturing agents (e.g., NP-40, sodium dodecyl
sulfate) may require adjustment of the salt concentration and the
temperature, as can readily be determined by one skilled in the
art.
[0039] Non-limiting examples of hybridization conditions that may
be used in the present invention are as follows. In Southern blot
analysis, the following hybridization conditions may be used: 30%
to 50% formamide in 2.times.SSC at 42.degree. C. After
hybridization, the filters are washed using standard methods. For
example, three 15 minute post-hybridization washes at 25.degree. C.
in 2.times.SSC to 0.1.times.SSC and 0.1% SDS may be carried out in
order to remove unbound probes. For RNA blots hybridizations in 30%
formamide at room temperature overnight were performed. Excess
probes were removed by washing three 15 min washes in 2.times.SSC
with 0.1% SDS.
[0040] The term "hybridization" refers to the pairing of
complementary nucleic acids. Hybridization and the strength of
hybridization (i.e., the strength of the association between the
nucleic acids) is impacted by such factors as the degree of
complementary between the nucleic acids, stringency of the
conditions involved, the T.sub.m of the formed hybrid, and the G-C
ratio within the nucleic acids. A single molecule that contains
pairing of complementary nucleic acids within its structure is said
to be "self-hybridized."
[0041] It is well known that numerous equivalent conditions may be
employed to comprise suitable hybridization conditions; factors
such as the length and nature (DNA, RNA, base composition) of the
probe and nature of the target (DNA, RNA, base composition, present
in solution or immobilized, etc.) and the concentration of the
salts and other components (e.g., the presence or absence of
formamide, dextran sulfate, polyethylene glycol) are considered and
the hybridization solution may be varied to generate conditions of
low stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions that
promote hybridization under conditions of high stringency (e.g.,
increasing the temperature of the hybridization and/or wash steps,
the use of formamide in the hybridization solution, etc.).
[0042] For in situ hybridization, the following conditions may be
used as described in U.S. Pat. No. 6,165,723 and U.S. patent
application Ser. No. 11/494,430 (which are herein incorporated by
reference): GuSCN (1.5 to 3.5 M depending on the probe sequence)
between room temperature and 37.degree. C. or mixtures of GuSCN and
formamide between room temperature and 37.degree. C. for 30 minutes
to one hour, followed by washes in SSC (2.times. to 0.1.times.) and
0.1%SDS.
[0043] Exemplification
[0044] Without further elaboration, it is believed that one skilled
in the art can, based on the description herein, utilize the
present invention to its fullest extent. The following specific
embodiments are, therefore, to be construed as merely illustrative,
and not limitative of the remainder of the disclosure in any way
whatsoever.
[0045] Hybridization of probes PGenus1, PGenus2, MalF1, MalF2,
Mal1.8F, Mal1.8R to P. Falciaprum, P. Vivax, P. Ovale and P.
Malariae Samples.
[0046] Dot-blot analysis data is shown in Table 1. In these
experiments, approximately 0.1 ug of a plasmid containing
nucleotide sequences encoding the respective 18S rRNA subunits was
used per spot. The blots were hybridized with dig-labeled probes
under the hybridization conditions described above.
[0047] In case of RNA, RNA was synthesized and 0.1 ug in
6.times.SSC was spotted on nitrocellulose membrane. Hybridization
with the dig-labeled probes (digoxigenin-labeled probes) was
performed overnight at room temperature in formamide. This method
was used to compare hybridization signals between the different
organisms and probes.
[0048] Probes which hybridize to all species of 18 rDNA of all
species of Plasmodium are PGenus1, PGenus2, MalF1, MalF2, Mal1.8F
and Mal1.8R. Probes PGenus1, PGenus2 and Mal1.8R hybridized to 18S
rRNA of all the Plasmodium species.
[0049] Plasmodium Genus 18S rRNA-Specific Probes
[0050] Plasmodium genus specific probes of the invention include
probes PGenus1, PGenus2, MalF1, MalF2, Mal1.8F and Mal1.8R which
have the sequences:
TABLE-US-00001 PGenus1 (SEQ ID NO: 1) 5'-TCTCGCTTGCGCGAATACTCG-3'
PGenus2 (SEQ ID NO: 2) 5'-CCAAAGACTTTGATTTCTCAT-3' Malf1 (SEQ ID
NO: 3) 5'-CAGATACCGTCGTAATCTTA-3' Malf2 (SEQ ID NO: 4)
5'-CGAAAGTTAAGGGAGTGAAGAC-3' Mal1.8F (SEQ ID NO: 5)
5'-atgtagaaactgcgaacggc-3' Mal1.8R (SEQ ID NO: 6)
5'-cagcacaatctgatgaatcatgc-3'
[0051] Plasmodium Species 18S rRNA-Specific Probes
[0052] Plasmodium species specific probes of the invention include
probes PV1 which have the sequences of a probe selected from PV1,
PV2, PV3, PF1, PF2, PF3, PF4, PF5, PF6 PF7, PF8, PM1, PO1 or the
full-length complementary sequence thereof.
TABLE-US-00002 P. vivax Specific Probes PV1 (SEQ ID No: 7)
5'-TCTAAGAATAAACTCCGAAGAGAAAATTCTTATTTT-3' PV2 (SEQ ID No: 8)
5'-TACACACTCAAGAAATGAATCAAGAGTGC-3'
TABLE-US-00003 P. falciparum Specific Probes PF1 (SEQ ID NO: 9)
5'-GCAATCTAAAAGTCACCTCGAAAGATGACTT-3' PF2 (SEQ ID No: 10)
5'-CCTAACAAATACTTATCCAAAGATAAAAATCAAGGA-3' PF3 (SEQ ID No: 11)
5'-ATTTTTAACACTTTCATCCAACACCTAGTCG-3' PF4 (SEQ ID No: 12)
5'-TTACAAAACCAAAAATTGGCCTTGCATTGTTATTT-3' PF5 (SEQ ID No: 13)
5'-TCCAATTGTTACTCTGGGAAGG-3' P. malariae Specific Probe PM1 (SEQ ID
No: 14) 5'-GAAACACTCATATATAAGAATGTCTC-3' P. ovale Specific Probe
PO1 (SEQ ID No: 15) 5'-AATTTCCCCGAAAGGAATTTTC-3'
TABLE-US-00004 TABLE 1 Probes P. falciparum P. vivax P. malariae P.
ovale PGenus1 Positive Positive Positive Positive PGenus2 Positive
Positive Positive Positive MalF1 Positive Positive Positive
Positive MalF2 Positive Positive Positive Positive Mal1.8F Positive
Positive Positive Positive Mal1.8R Positive Positive Positive
Positive PV1 Negative Positive Negative Negative PV2 Negative
Positive Negative Negative PF1 Positive Negative Negative Negative
PF2 Positive Negative Negative Negative PF3 Positive Negative
Negative Negative PF4 Positive Negative Negative Negative PF5
Positive Negative Negative Negative PM1 Positive Negative Positive
Negative PO1 Positive Negative Negative Positive Note: All the
probes except MalF1, MalF2 and Mal1.8F hybridize to rRNA also.
[0053] In one exemplification of the present invention, samples are
tested for the presence of Plasmodium sp. and detected Plasmodium
species are differentiated by use of the probes of the present
invention. A sample is tested with probes of at least five, ten or
fifteen contiguous nucleotides of probes PV1 and/or PV2, probes
PF1, PF2, PF3, PF4 and/or PF5, probes PM1 and probe PO1, or the
entire probe or the complementary sequences thereof. Samples that
test positive for probes PV1 and/or PV2 are determined to be
infected with P. vivax. Samples that test positive for probes PF1,
PF2, PF3, PF4 and/or PF5 are determined to be infected with P.
falciparium. Samples that test positive for probes PM1 but not for
PF1, PF2, PF3, PF4 and/or PF5 are determined to be infected with P.
malariae. Samples that test positive for probes PO1 but not for
PF1, PF2, PF3, PF4 and/or PF5 are determined to be infected with P.
ovale.
[0054] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention and, without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
Sequence CWU 1
1
15121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 1tctcgcttgc gcgaatactc g 21221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
2ccaaagactt tgatttctca t 21320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 3cagataccgt cgtaatctta
20422DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 4cgaaagttaa gggagtgaag ac 22520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
5atgtagaaac tgcgaacggc 20623DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 6cagcacaatc tgatgaatca tgc
23736DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 7tctaagaata aactccgaag agaaaattct tatttt
36829DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 8tacacactca agaaatgaat caagagtgc 29931DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
9gcaatctaaa agtcacctcg aaagatgact t 311036DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
10cctaacaaat acttatccaa agataaaaat caagga 361131DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
11atttttaaca ctttcatcca acacctagtc g 311235DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
12ttacaaaacc aaaaattggc cttgcattgt tattt 351322DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
13tccaattgtt actctgggaa gg 221426DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 14gaaacactca tatataagaa
tgtctc 261522DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 15aatttccccg aaaggaattt tc 22
* * * * *